Organization of the ribosomal RNA gene cluster in Aspergillus nidulans

DNA coding for ribosomal RNA in Aspergillus nidulans was found to consist of a unit 7.8 kb in size which is tandemly repeated in the genome and codes for 5.8S, 18S and 26S rRNA. The repeat unit has been cloned, and its restriction map and the location of the individual rRNA coding sequences within the unit have been established.     

Cloning and characterization of the aldA gene of Aspergillus nidulans

We have cloned and sequenced the aldA (encoding aldehyde dehydrogenase) gene of Aspergillus nidulans. The gene contains two introns which are similar in size and structure to other fungal introns. The amino acid sequence of aldehyde dehydrogenase (497 residues) shows a significant level of homology with analogous sequences in other organisms. Comparison of the primary structure of the active sites of the mammalian cytosolic and mitochondrial enzymes shows that the Aspergillus enzyme closely resembles the mammalian mitochondrial enzyme. Analysis of the 5' non-coding region of the aldA gene shows a TATA-like sequence located 90 bp upstream from the initiation codon. Two messenger-RNA start points are located 36 and 42 bp upstream from the start codon.     

Metabolite repression and inducer exclusion in the proline utilization gene cluster of Aspergillus nidulans

The clustered prnB, prnC, and prnD genes are repressed by the simultaneous presence of glucose and ammonium. A derepressed mutation inactivating a CreA-binding site acts in cis only on the permease gene (prnB) while derepression of prnD and prnC is largely the result of reversal of inducer exclusion.     

Identification of a gene for beta-tubulin in Aspergillus nidulans.

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.     

Identification of a gene for alpha-tubulin in Aspergillus nidulans.

This paper demonstrates that revertants of temperature-sensitive benA (β-tubulin) mutations in Aspergillus nidulans can be used to identify proteins which interact with β-tubulin. Three benomyl-resistant benA (β-tubulin) mutants of Aspergillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts^?) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts⁺ were isolated by selection at the restrictive temperature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had β-tubulins which differed from the β-tubulins of their parental strains by one (1^?) or two (2^?) negative charges on two-dimensional gel electrophoresis. Although the β-tubulins of the extragenic suppressor strains were all electrophoretically identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in α1-tubulin (1⁺). A heterozygous diploid between this strain and a strain with wild-type α1-tubulin was found to have both wild-type and mutant (1⁺) α1-tubulins. This experiment rules out post-translational modification as a possible cause of the α1-tubulin abnormality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for α1-tubulin. We propose that this gene be designated tubA to denote that it is a gene for α1-tubulin in A. nidulans.     

Cloning and characterization of the gene encoding Aspergillus nidulans DNA topoisomerase II.

We have determined the complete nucleotide sequence of a 5544bp genomic DNA fragment from Aspergillus nidulans that encodes DNA topoisomerase II (topo II). It contains a single open reading frame of 4740bp that codes for 1579 amino acid residues with a molecular weight of 178kDa; when expressed in Escherichia coli and Saccharomyces cerevisiae the molecular weight was 180kDa. The gene (TOP2) is divided into three exons. Two introns, 54bp and 60bp in length, are located at nucleotide positions 187 and 3214 respectively. Comparison of the deduced amino acid sequence with other eukaryotic topo II sequences showed a higher degree of identity with other fungal enzymes than the human topo IIalpha. One of monoclonal antibodies raised against human topo II, 6H8, can cross-react with Aspergillus topo II.     

Bidirectional gene transfer between Aspergillus fumigatus and Aspergillus nidulans

Abstract The rpmF-plsX-fabH gene cluster of Rhodobacter capsulatus homologous to that of Escherichia coli was identified. rpmF encodes ribosomal protein L32, plsX plays an undefined role in membrane lipid synthesis, and fabH encodes β-ketoacyl-acyl carrier protein synthase III. The R. capsulatus plsX gene complemented a defect in an E. coli strain with the plsX50 mutation. Overproduction of the fabH gene product of R. capsulatus in E. coli resulted in dramatically increased β-ketoacyl-acyl carrier protein synthase III activity. These results indicate that plsX and fabH apparently function the same in R. capsulatus as in E. coli .     

Genetic regulation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans

A large number of quinic acid non-utilizing qut mutants of Aspergillus nidulans deficient in the induction of all three quinic acid specific enzymes have been analysed. One class the qutD mutants, are all recessive and are non-inducible at pH 6.5 due to inferred deficiency in a quinate ion permease. Two regulatory genes have been identified. The QUTA gene encodes an activator protein since most qutA mutants are recessive and non-inducible although a few fully dominant mutants have been found. The QUTR gene encodes a repressor protein since recessive mutations are constitutive for all three enzyme activities. Rare dominant non-inducible mutants which revert readily to yield a high proportion of constitutive strains are inferred to be qutR mutants defective in binding the inducer. The gene cluster has been mapped in the right arm of chromosome VIII in the order: centromere - greater than 50 map units - ornB - 12 map units - qutC (dehydratase)-0.8 map units-qutD (permease), qutB (dehydrogenase), qutE (dehydroquinase), qutA (activator)-4.4 map units - qutR (repressor)-20 map units - galG. This organization differs from that of the qa gene cluster in Neurospora crassa, particularly in the displacement of qutC and qutR.     

Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans

Summary The functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain. The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase). The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa. The QUTD gene shows strong homology with the N. crassa QA-Y gene and QUTG with the QA-X gene. QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5 non-coding regions show significant homology with UAS _GAL and UAS _QA sequences of the Saccharomyces cerevisiae and N. crassa Gal and QA systems. In addition, conserved 5 sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented.